Enzyme characterization and biological activities of a resuscitation promoting factor from an oil degrading bacterium Rhodococcus erythropolis KB1
Enzyme characterization and biological activities of a resuscitation promoting factor from an oil degrading bacterium Rhodococcus erythropolis KB1
复制标题
油降解菌红平红球菌 KB1 复苏促进因子的酶特性和生物活性
DOI：
10.7717/peerj.6951
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发表时间：
2019-05-21
期刊：
影响因子：
2.7
通讯作者：
Wang, Yonggang
中科院分区：
文献类型：
--
作者：
Luo, Dan;Chen, Jixiang;Wang, Yonggang
复苏促进因子（resuscivation -promoting factors, Rpf）是一类水解酶，在不良培养基中参与休眠细胞的恢复和促进细菌生长。本研究构建了油降解细菌红红红球菌KB1中rrf -1基因的表达载体，并在大肠杆菌中表达。表达的蛋白经Ni2+亲和层析纯化，以4- methylumbellliferyl -β-D-N，N '，N″-triacetyl chitotrio苷为底物测定其水解活性。将纯化的Rpf-1添加到红血球培养物中，可以有效地促进细菌细胞的生长。纯化后的蛋白还能使红血球有活力但不可培养的细胞复苏到可培养状态。Rpf-1的保守氨基酸残基包括Asp45、Glu51、Cys50、Thr60、Gln69、Thr74、Trp75和Cys114被不同的氨基酸取代。用Ni2+亲和层析对突变蛋白进行了表达和纯化。与野生型Rpf-1相比，突变蛋白的水解活性有不同程度的下降。Gln69在酶活性中起着最重要的作用，用赖氨酸（Q69K）取代Gln69导致酶活性下降最大。其他氨基酸残基如Asp45、Glu51、Cys50和Cys114对维持Rpf-1的水解活性和生物学功能也非常重要。结果表明，红血球Rpf-1具有水解活性和较弱的蛋白酶活性，但其促生长和复苏活性是由水解活性决定的。
Resuscitation-promoting factors (Rpf) are a class of muralytic enzymes, which participate in recovery of dormant cells and promoting bacteria growth in poor media. In the present study the expression vector of the rpf-1 gene from an oil-degrading bacterium Rhodococcus erythropolis KB1 was constructed and expressed in Escherichia coli. The expressed protein was purified by Ni2+-affinity chromatography, and showed muralytic activity when measured with 4-methylumbelliferyl-β-D-N,N′,N″-triacetyl chitotrioside as substrate. Addition of purified Rpf-1 to R. erythropolis culture efficiently improved bacterial cell growth. The purified protein also increased resuscitation of viable but nonculturable cells of R. erythropolis to culturable state. The conserved amino acid residues including Asp45, Glu51, Cys50, Thr60, Gln69, Thr74, Trp75 and Cys114 of the Rpf-1 were replaced with different amino acids. The mutant proteins were also expressed and purified with Ni2+-affinity chromatography. The muralytic activities of the mutant proteins decreased to different extents when compared with that of the wild type Rpf-1. Gln69 was found to play the most important role in the enzyme activity, substitution of Gln69 with lysine (Q69K) resulted in the greatest decrease of muralytic activity. The other amino acid residues such as Asp45, Glu51, Cys50 and Cys114 were also found to be very important in maintaining muralytic activity and biological function of the Rpf-1. Our results indicated that Rpf-1 from R. erythropolis showed muralytic activities and weak protease activity, but the muralytic activity was responsible for its growth promotion and resuscitation activity.